Liver histology is associated with long-term clinical outcomes in patients with metabolic dysfunction–associated steatohepatitis

Background: Few studies have examined the risk of long-term clinical outcomes in patients with metabolic dysfunction–associated steatohepatitis in relation to liver histology. We aimed to study this using a real-world cohort. Methods: Adults (N = 702) recorded on Vanderbilt University Medical Center’s Synthetic Derivative database (1984–2021) with evidence of metabolic dysfunction–associated steatohepatitis on liver biopsy were followed from the first biopsy until the first clinical event or last database entry (median: 4.7 y). Risks of cirrhosis (N = 650), other noncirrhotic liver-related (N = 702) and cardiovascular-related outcomes (N = 660), and mortality due to liver, cardiovascular, or cancer events (N = 660) were determined as a function of baseline histology (fibrosis stage [F], lobular inflammation grade [LI], hepatocyte ballooning grade [HB], and steatosis score) adjusting for sex, age, diabetes, and weight-loss surgery. Results: Cirrhosis risk was reduced for lower versus higher fibrosis stage (HR: F0–1 vs. F3: 0.22 [95% CI: 0.12–0.42]), LI1 versus LI2–3 (0.42 [0.19–0.97]), and HB1 versus HB2 (0.20 [0.08–0.50]). Lower fibrosis stage was associated with significantly lower risks of liver-related outcomes versus F4 cirrhosis (eg, F0–1: 0.12 [0.05–0.25]), whereas no differences were seen across baseline lobular inflammation, hepatocyte ballooning, and steatosis grades/scores. Lower versus higher lobular inflammation grade was associated with lower risks for liver-related outcomes in patients with weight-loss surgery. There was a trend for lower risks for cardiovascular-related and any long-term outcomes with lower versus higher fibrosis stage. Conclusions: Fibrosis stage and lobular inflammation and hepatocyte ballooning grades predict the risk of long-term outcomes, supporting the use of these histological features as potential surrogate markers of disease progression or clinical outcomes.


Biopsy interpretation assumptions:
• Standard scale for lobular inflammation and steatosis is mild/moderate/severe (or marked) • If range is given (e.g.mild to moderate), then scoring based on more severe end of range • Negative for/no steatohepatitis = no lobular inflammation or hepatocyte ballooning • Minimal/focal/rare/minute/small/little understood to mean mild/few across all scales • Diffuse understood to mean severe (or marked) • Pericellular/perisinusoidal/perivenular considered roughly equivalent terms 1.Any biopsy with at least 5% steatosis will be considered for inclusion • Biopsies with no steatosis are excluded (biopsies with no steatosis but high fibrosis could be considered as possible end-stage MASH) • If steatohepatitis or MASLD/NAFLD is mentioned in the report, then biopsy is included regardless of amount of steatosis 2. Any biopsy with obvious mention of other causes of steatosis or steatohepatitis will be excluded (e.g.methotrexate use, Wilson disease, etc.) 3. Any biopsy mentioning "steatohepatitis" will be placed in the more likely MASH category • Any biopsy mentioning "borderline steatohepatitis" will be placed in the more likely

MASH category
• Any biopsy mentioning "not steatohepatitis" or equivalent will be placed in the more likely MASL category 4. Any biopsy with NAS score of 5 or higher will be placed in the more likely MASH category 5. Any biopsy (with NAS score of less than 5) demonstrating both hepatocyte ballooning degeneration and pericellular/perisinusoidal fibrosis will be placed in the more likely MASH category (biopsies with only pericellular/perisinusoidal fibrosis or only hepatocyte ballooning degeneration could be considered as more likely MASH) 6.Any biopsy with evidence of severe fibrosis/cirrhosis will be placed in the more likely MASH category 7. Any biopsy lacking features 3-6 will be placed in the more likely MASL category

General considerations
If not otherwise specified, the main analysis and Fibrosis-4 index (FIB-4) analysis were done on all patients in the analysis set, whereas analyses for cirrhosis events were done on the subpopulation with fibrosis stage F0 to F3 at baseline and no cirrhosis events prior to the index date.All statistical analyses were performed using R version 4.0.3, and the R packages survival 3.2-7, survminer 0.4.9, xtable 1.8-4, and Hmisc 4.4.2.

Significance level
A significance level of 0.05 for 2-sided comparisons was considered statistically significant, for example, p<0.05 in log-rank test.HRs and 95% CIs were derived.The differences in time to events were also treated as statistically significant if 95% CIs did not cross one.

Presentation of continuous and qualitative variables
Descriptive statistics derived for continuous variables (e.g., age and BMI) were reported as median (IQR).Qualitative variables were summarized by counts and percentages based on the number of patients in the analysis of interest, unless otherwise specified.

Definition of index event
The date of the first liver biopsy pathology report with histologic evidence of MASH was used as the index event for each patient.Fibrosis stage, total NAS score, and the NAS score subcomponents (lobular inflammation, hepatocyte ballooning, steatosis) at baseline were defined using the information documented in the pathology report and according to the review criteria outlined in the histology evaluation criteria section above.

Definition of event
If not otherwise specified, patient records were surveyed from the date of the index event until the most recent EMR data entry to identify the first incidence of a clinical event of interest.
This was based on the assumption that the patient did not have a pre-existing diagnosis of the clinical outcome prior to receiving health care within the Vanderbilt University Medical Center system.The validity of this assumption was checked for each event by evaluating the number of events in close proximity to the index date and excluding patients with pre-existing conditions accordingly (e.g., with the first event incidence occurring within 30 days of the index date).
The time to event (i.e., time to first diagnosis) was calculated as event date minus index date.
For the analysis focusing on change in FIB-4 from baseline to 360 days (90 days) follow-up, time to event was calculated as event date minus the date 30 days past the date of the 360-day FIB-4 follow-up.

Main analyses
Patients with pre-existing cardiovascular events prior to the liver biopsy index date were excluded from analysis of both cardiovascular-related events and overall diagnostic incidence.
In the main analysis, death from any cause was considered as a competing event of any nonfatal event.The censoring observations caused by deaths happening before the non-fatal events were not assumed to be independent of the event time.The Aalen-Johansen method with death as a competing risk was used to estimate the cumulative probabilities of non-fatal events for each subtype of fibrosis stages and baseline liver-related histological parameters (the cumulative probability curves with zero events were not plotted), and the Fine-Gray competing risk model was used to estimate the sub-distribution hazard ratios between subtypes.
For the total outcome event, the Cox proportional hazards model was used, where the mortality status at end of follow-up was treated as censored rather than missing if no mortality occurred during follow-up.The score (log rank) test was used to compare the hazards between subtypes, and the p-value presented to show significance (if p < 0.05).Also, the HRs without and with adjustment for covariates were shown, denoted as the crude HRs and the adjusted HRs, respectively, along with the 95% CIs.The covariates used in Fine-Gray competing risk or Cox models included baseline age, gender, baseline diabetes, and baseline weight loss surgery.In the analyses for cirrhosis, HCC, MELD score, and decompensation events, one case with liver transplant was excluded.
Using similar statistical methodology to the main analyses, HRs and 95% CIs were calculated with covariates as change in FIB-4 from baseline, age at baseline, gender, baseline diabetes status, weight loss surgery status at baseline and respective FIB-4 value at baseline (baseline refers to the index date defined as first identified biopsy).Results from the multivariable modelling were also used to understand the effect on outcomes by other covariates.

Missing data and imputation rules
In the analysis of association of time to events with NAS score, which is the summation of three baseline liver-related histological parameters (steatosis, lobular inflammation, and hepatocyte ballooning), multiple imputation (with five imputations) for missing values of baseline liverrelated histological parameters was performed through additive regression, bootstrapping, and predictive mean matching.The imputation did not include missing time-to-outcome and censored status.The final results were the average of the five imputed datasets.The multiple imputation approach took all aspects of uncertainty of the imputations into account by using bootstrap to approximate the process of drawing predicted values from a full Bayesian (posterior) predictive distribution.After imputations, the Fine-Gray competing risk or Cox model was applied to each of the five imputed datasets.The analysis results were different for each imputed dataset due to the randomness of the predicted values, and there was a variance between the results.Incorporating the results with the variance, the average results were reported, along with the 95% CIs.

FIB-4 analyses
The baseline measurement (including FIB-4 index) was defined as the measurement occurring nearest in time to the index date within a ± 90-day window.An exception was made for laboratory findings with multiple values on the same day in which case the baseline value is defined as the mean of the available measurements.The time in years from 30 days after the date of the second FIB-4 measurement to the date of a liver-related, cardiovascular-related, or any long-term outcome was calculated, excluding patients with cardiovascular events prior to the liver biopsy index date from analysis of both cardiovascular-related events and overall outcomes.For this analysis, the Fine-Gray competing risk model was used to investigate the association of the time to an outcome event with changes in FIB-4, defined as the difference in value at 360 days (90 days) follow-up versus baseline.Patients with clinical outcome of interest or lost to follow-up between baseline and 30 days past the date of 360-day FIB-4 follow-up were excluded from the analysis.Crude HRs and adjusted HRs were reported.Covariates included baseline FIB-4 value, baseline age, gender, baseline diabetes, and baseline weight loss surgery.No time-varying variable was used in regression models.Liver-related outcomes were defined as any of the following: HCC, MELD ≥15, hepatic decompensation, liver transplant, or liver-related mortality.Cirrhosis was analysed separately in patients with F0-3 at baseline and not included in liver-related outcomes.Cardiovascular-related outcomes included any of the following:

List of ICD and CPT codes
heart failure, myocardial infarction, ischemic stroke, unstable angina, and coronary revascularization, and cardiovascular-related mortality for patients with no cardiovascular outcome events at baseline.Any long-term outcome was defined as any liver-related [excluding cirrhosis], and cardiovascular-related outcomes, and mortality due to liver, cardiovascular or cancer events.Abbreviations: CI, confidence interval; FIB-4, fibrosis-4 index; HR, hazard ratio.

•
Septal fibrosis mentioned by itself = portal extension• Activity = lobular inflammation (but must specify lobular activity or be associated with description of steatohepatitis)• If hepatitis grade reported, then 1-2 = minimal-mild, 3 = moderate, 4 = severe • No hepatitis = no inflammation • Steatosis without active…/without activity… = no lobular inflammation or hepatocyte ballooning • No hepatocyte injury/hepatocytes without damage = no hepatocyte ballooning • No significant histopathologic change (or equivalent) = no abnormalities for any finding not otherwise addressed Biopsy interpretation qualifier key: i -Parameter not reported and no further clinical determination possible ii -Parameter not reported but clinical inference possible iii -Parameter reported but not sufficiently described to directly apply to a scale -clinical judgement required • Steatosis not mentioned but steatohepatitis is = steatosis of 1 with qualifier of iii iv -Parameter reported but described in a non-standardized fashion -clinical judgement required • Steatosis: patchy = mild, scattered = moderate • lobular inflammation: spotty/patchy = mild, occasional/scattered = moderate • hepatocyte ballooning: occasional/scattered/some/spotty = few, frequent/moderate/well-developed = many • Zone 3 fibrosis: spotty/patchy/occasional/scattered = mild Biopsy categorization (more likely MASH vs. more likely MASL):

TABLE 1 .
Additional baseline characteristics (NAS and components) stratified by baseline stages of fibrosis.
Abbreviations: F, fibrosis stage; N, number of patients total; n, number of patients with each grade/score; NAS, non-alcoholic fatty liver disease activity score.SDC

TABLE 2 .
Key baseline characteristics stratified by baseline grades of hepatocyte ballooning.

TABLE 3 .
Key baseline characteristics stratified by baseline grades of lobular inflammation.

TABLE 4 .
Key baseline characteristics stratified by baseline scores of steatosis.

TABLE 5 .
Hazard ratios and incidence rates for liver-related outcomes, incident cirrhosis, and cardiovascular-related outcomes, stratified by baseline fibrosis stage (overall and individual events).

years at risk Incidence rate per 100 person-years HR (95% CI), p value
total number of patients at risk.All HRs were adjusted for sex, baseline age, diabetes, and weight loss surgery status, unless otherwise specified.Liver-related outcomes were defined as any of the following: HCC, MELD ≥15, hepatic decompensation, liver transplant, or liver-related mortality.Cirrhosis was analysed separately in patients with F0-3 at baseline and not included in liver-related outcomes.Cardiovascular-related outcomes included any of the following: heart failure, myocardial infarction, ischemic stroke, unstable angina, and coronary revascularization, and cardiovascular-related mortality for patients with no cardiovascular outcome events at baseline.Any long-term outcome was defined as any liver-related [excluding cirrhosis], and cardiovascular-related outcomes, and mortality due to liver, cardiovascular or cancer events.

TABLE 6 .
Hazard ratios and incidence rates for liver-related outcomes, incident cirrhosis, and cardiovascular-related outcomes, stratified by baseline hepatocyte ballooning grade (overall and individual events).totalpatients at risk.All HRs were adjusted for sex, baseline age, diabetes, and weight loss surgery status, unless otherwise specified.Liver-related outcomes were defined as any of the following: HCC, MELD ≥15, hepatic decompensation, liver transplant, or liver-related mortality.Cirrhosis was analysed separately in patients with F0-3 at baseline and not included in liver-related outcomes.Cardiovascular-related outcomes included any of the following: heart failure, myocardial infarction, ischemic stroke, unstable angina, and coronary revascularization, and cardiovascular-related mortality for patients with no cardiovascular outcome events at baseline.Any long-term outcome was defined as any liver-related [excluding cirrhosis], and cardiovascular-related outcomes, and mortality due to liver, cardiovascular or cancer events.

TABLE 7 .
Hazard ratios and incidence rates for liver-related outcomes, incident cirrhosis, and cardiovascular-related outcomes, stratified by baseline lobular inflammation grade (overall and individual events).total patients at risk.All HRs were adjusted for sex, baseline age, diabetes, and weight loss surgery status, unless otherwise specified.

TABLE 8 .
Abbreviations: CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; LI, lobular inflammation; MELD, model for end-stage liver disease; NC, not calculated; REF, reference comparator.Exploratory analysis of FIB-4: adjusted hazard ratios for liver-related outcomes.Follow-up value extracted 360 days (±90 days) from the patient's index date (date of the first MASH liver biopsy).Patients with liver-related event before the second FIB-4 measurement date +30 days were excluded. SDC